Systems for the detection of target sequences

Abstract

The present invention relates to means for cleaving a nucleic acid cleavage structure in a site-specific manner. Enzymes, including 5′ nucleases and 3′ exonucleases, are used to detect and identify nucleic acids derived from microorganisms. Methods are provided which allow for the detection and identification of bacterial and viral pathogens in a sample.

1. A composition, comprising
 a) a target nucleic acid, said target nucleic acid comprising a first region and a second region, said second region downstream of said first region;  
 b) a first oligonucleotide comprising a 3′ portion and a 5′ portion;  
 c) a second oligonucleotide; and  
 d) a third oligonucleotide comprising a hairpin structure comprising a duplex region and a single-stranded region;  
 wherein at least said 3′ portion of said first oligonucleotide is complementary to said first region of said target nucleic acid, wherein at least a portion of said second oligonucleotide is complementary to said second region of said target nucleic acid, and wherein said 5′ portion of said first oligonucleotide is complementary to at least a portion of said third oligonucleotide.  
 
     
     
       2. The composition of  claim 1 , wherein said third oligonucleotide comprises a label. 
     
     
       3. The composition of  claim 2 , wherein said label comprises a fluorophore. 
     
     
       4. The composition of  claim 2 , wherein said label comprises a fluorophore having quenched emission. 
     
     
       5. The composition of  claim 2 , wherein said label is selected from the group consisting of biotin, a radiolabeled, a hapten, a detectable nucleic acid, a peptide, a chromophore, and a dye. 
     
     
       6. The composition of  claim 1 , further comprising a solid support attached to at least one of said first, second and third oligonucleotides. 
     
     
       7. The composition of  claim 6 , wherein said first oligonucleotide is attached to said solid support. 
     
     
       8. The composition of  claim 6 , wherein said second oligonucleotide is attached to said solid support. 
     
     
       9. The composition of  claim 6 , wherein said third oligonucleotide is attached to said solid support. 
     
     
       10. The composition of  claim 1 , wherein said first and said second regions of said target nucleic acid are adjacent to each other. 
     
     
       11. The composition of  claim 1 , wherein said portion of said second oligonucleotide complementary to said second region of said target nucleic acid comprises a 3′ terminus. 
     
     
       12. The composition of  claim 1 , wherein said single stranded region of said hairpin structure of third oligonucleotide comprises a single-stranded 3′ arm adjacent to said duplex region of said hairpin structure. 
     
     
       13. The composition of  claim 12 , wherein said portion of said third oligonucleotide that is complementary to said 5′ portion of said first oligonucleotide comprises at least a region of said single-stranded 3′ arm of said hairpin structure. 
     
     
       14. The composition of  claim 13 , wherein said region of said single-stranded 3′ arm is adjacent to said duplex region of said hairpin structure.